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Colorado State University College of Veterinary Medicine and Biomedical Sciences College of Veterinary Medicine and Biomedical Sciences
Department of Biomedical Sciences

Rash Laboratory

Journal of Neurocytology 33, 131–151 (2004)

High-resolution proteomic mapping in the vertebrate central nervous system:

Close proximity of connexin35 to NMDA glutamate receptor clusters and

co-localization of connexin36 with immunoreactivity for zonula occludens

protein-1 (ZO-1).

[click on images for a larger view]

Fig. 1 Confocal images of Mauthner cells labeled for Cx35 and glutamate receptor NR1, with companion FRIL immunogold labeling of gap junctions in goldfish Mauthner cells.

(A) Confocal immunofluorescence image (three Z sections of 2 µm) showing Cx35 (MAB 3045) at a Club Ending on distal portion of a Mauthner Cell lateral dendrite. These terminals, identified by their large size, exhibit multiple sites of punctate labeling for Cx35. (B and C) Cx35 (Ab298) labeling of conventional “plaque” gap junctions (red overlay) in MC/CE synapses. Both images are from the same goldfish MC/CE synapse, showing primarily postsynaptic P-face (B) and presynaptic E-face (C). In both images, all labeling is on postsynaptic connexins. (D) Laser scanning immunofluore-scence image (three Z sections of 2 µm) of a Club Ending on a Mauthner-cell lateral dendrite after labeling with anti-NR1 antibody. (E–H) Simultaneous co-localization of Cx35 at gap junctions and NR1 glutamate receptors in E-face particles arrays (yellow overlay) in goldfish Mauthner cell. (E) E-face view of the postsynaptic membrane of goldfish Mauthner cell. E-face image of gap junction in postsynaptic membrane, but with unfractured and unreplicated presynaptic plasma membrane beneath. It is these unreplicated presynaptic connexins that are labeled (18 nm gold). In contrast, the two arrays of E-face IMPs represent glutamate receptor clusters, which are immunogold labeled for NR1 (12 nm gold beads in the extracellular space; see Fig. 2B–E for explanatory diagram). Inscribed areas are shown at higher magnification (F–H). (F) Stereoscopic (left pair) and reverse stereoscopic images (right pair) of a neuronal gap junction labeled for Cx35. (G and H) Portions of two PSDs after immunogold labeling for glutamate receptor NR1 by two gold beads (G) and one gold bead (H). The gap junction and the PSD on the right are separated by~50 nm, or less than the limit of resolution of light microscopy. For confocal images A and D, calibration bars = 5 µm. In all FRIL replicas, calibration bars = 0.1 µm, unless otherwise designated.

Fig. 2 Immunogold labeling of glutamate receptor PSDs and nearby gap junctions.

(A) Portions of three Cx35-immunogoldlabeled gap junctions (red overlays) surrounding a P-face imprint of a PSD (yellow overlay), corresponding to the arrays of IMPs on E-faces (see Fig. 1G and H). The PSD P-face pits are devoid of NR-1 labeling. (B–E) Drawing of fracture plane through two Club Ending synapses on a Mauthner cell. (B and C) From left to right, the fracture plane (blue line) sequentially fractures within the postsynaptic plasma membrane, through a glutamate receptor PSD (lavender) and through a postsynaptic
gap junction (left side of image), across the Mauthner cell cytoplasm (center of image), then to within the Mauthner cell plasma membrane (right side), exposing the postsynaptic membrane E-face particles corresponding to glutamate receptors, then the gap junction E-face pits (postsynaptic membrane) and, finally, gap junction P-face particles (presynaptic membrane of lower club ending). (B and C) PSD P-face pits are not labeled because no glutamate receptor proteins remain with the P-face pits (left side). However, P-face connexon IMPs (orange connexons, left side) in the adjacent gap junction are labeled. Glutamate receptor E-face particles are labeled on their extracellular determinants (right side), whereas connexons of presynaptic gap
junctions are labeled on their cytoplasmic epitopes (D and E). In the gap junction on the right (2C, box) and in closer view (D), postsynaptic E-face pits and presynaptic P-face particles are replicated, but in both cases, it is the presynaptic connexons (yellow connexons) of the underlying Club Ending that are labeled. (E) Diagram showing immunogold labeling of epitope in
the cytoplasmic carboxy terminus of a single connexin molecule by Ab298 (Pereda et al., 2003). (F) Stereoscopic and reverse stereoscopic image of portion of mixed synapse in adult rat inferior olive labeled for Cx36 (20 nm gold; red overlay) and NR1 glutamate receptors (10 nm gold, arrow; yellow overlay). Reverse stereoscopy (right pair) is helpful for discerning the 10 nm gold bead superimposed on the equally electron-dense platinum replica, as well as for discriminating between the two layers of replica that arose when a portion of replica was displaced (darker area at top).

Fig. 3  FRIL images of "plaque", "string" and "ribbon gap junctions (red overlays) in inner and outer plexiform layers of adult rat retina after immunogold labeling for Cx36. (Criteria for identifying gap junctions are at the end of Materials and Methods.) (A) Two small, regular plaque gap junctions labeled for Cx36 (18nm gold beads). (B) Two single-strand "string" gap junctions, with both P-face particles and E-face pits occurring primarily as single rows of particles or pits. Immunogold beads are associated with most of t he connexon strands. (C) Immunogold-labeled multi-strand "ribbon" gap junction. Each "ribbon" is 2-6 IMPs (or pits) wide, with up to 500 connexons per compound ribbon gap junction. (D) One plaque-type gap junction consisting of ~12 pits (upper left) and labeled by four 12 nm immunogold beads, as well as one simple, single-strand ribbon gap junction (lower right) labeled by three 12 nm gold beads. Due to their small diameter, neither of these two gap junctions would be reliably resolved in a conventional thin section TEM image.


Fig. 4  Expression of Cx36 in the reticular thalamic nucleus (RTN) and co-localization with ZO-1 in 16-day -old mouse brains. (A) Low magnification double immunofluorescence showing punctate labeling of Cx36 (A1) and, in the same field, more widely distributed punctate labeling of ZO-1 (A2) in the RTN (outlined by dashed line) of a wildtype (WT) mouse, with substantial Cx36/ZO-1 co-localization shown by yellow in overlay of images (A3). (B) Low magnification double immunofluorescence images of a Cx36 knockout (KO) mouse. A field corresponding to that in A shows an absence of specific labeling for Cx36 (B1)and no discernible loss of specific labeling for ZO-1 (B2). Overlay (B3) shows reduced yellow in the RTN (outlined by dashed line). Residual green and red fluorescence in thalamic areas of Cx36 KO mice represents non-specific attachment of primary and secondary antibodies. Bright, threadlike labeling for ZO-1 is associated with blood vessels. (C) Laser scanning confocal double immunofluorescence showing magnification of Cx36 (C1) and ZO-1 (C2) co-localization (yeallow in overlay images, (C3) in a field of RTN in a WT mouse. Scale bars: A and B, 100 mm; C, 10 mm.

Fig. 5  Expression of Cx36 in the amygdala and Cx36 co-localization with ZO-1 in adult mouse brains. (A) Fluorescence Nissl-stained section showing the basomedial (BMA) and basolateral (BLA) nuclei of the amygdala (dashed lines). (B–D) Immunofluorescence labeling of Cx36 showing a dense concentration of Cx36-positive puncta in the BMA (B) of wildtype (WT)
mouse, much sparser distribution of Cx36-positive puncta in the BLA of WT mouse (C), and an absence of punctate labeling for Cx36 in the BMA of a Cx36 knockout (KO) mouse (D). (E, F) Low magnification (E) and higher magnification confocal (F) double immunofluorescence showing labeling of Cx36 (E1, F1) and, in the same fields, labeling of ZO-1 (E2, F2, respectively)
in the BMA of WT mouse, with Cx36/ZO-1 co-localization seen as yellow in overlay of images (E3, F3). Punctate labeling for ZO-1 is much denser than that for Cx36, and some intense ZO-1 labeling is seen in association with blood vessels (E2). Scale bars: A, 200 µm; B–D, 100 µm; C, 10 µm.

Fig. 6  Simultaneous labeling of Cx32 in gap junctions and ZO-1 in tight junctions of rat liver, and comparison with ZO-1 labeling in tight junctions of capillary in adult rat retina. (A) Stereoscopic imaging (left pair) and reverse stereoscopic imaging (right pair) reveals that Cx32 labeling (12 nm gold) is directly associated with gap junctions (white arrowheads), whereas labeling
for ZO-1 (18 nm beads) is associated exclusively with tight junctions strands (black arrowheads). (B) Portions of five capillary endothelial cells linked by tight junctions (arrow), each immunogold labeled for ZO-1 (12 nm gold beads). Asterisk (∗) denotes capillary lumen. (C) Stereoscopic and reverse stereoscopic images of tight junctions (arrows), with ZO-1 labeling beneath the

Fig. 7 (A–C) Simultaneous co-localization of Cx36 (6 nm gold beads [arrows] and 12 nm gold beads) and ZO-1 (18 nm gold beads) in “plaque” gap junctions linking unidentified neurons in adult rat retina. (C) Reverse stereoscopy facilitates detection of the smallest gold beads (Fig. 7B, right pair of images), as well as the “sidedness” of the labels. (C) Neuronal gap junction with seven 18 nm gold beads (ZO-1) and four 12 nm gold beads (Cx36).

Journal Neurocytology 2004

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